Method for producing cercosporamide

ABSTRACT

The present invention provides a process for producing cercosporamide comprising culturing a fungus belonging to the genus  Lachnum  and/or fungus belonging to the genus  Pseudaegerita  and recovering cercosporamide from the culture thereof. In addition, the present invention provides cercosporamide acquired according to this production process. Further, the present invention provides novel microorganisms consisting of  Lachnum fuscescens  SANK 19096,  Lachnum calycioides  SANK 12497,  Lachnum caesaliatum  SANK 10906, and  Pseudaegerita websteri  SANK 11006.

TECHNICAL FIELD

The present invention relates to a process for producing cercosporamideusing a fungus belonging to the genus Lachnum and/or belonging to thegenus Pseudaegerita, cercosporamide produced according to saidproduction process, and a fungus and the like that producescercosporamide.

BACKGROUND ART

Cercosporamide is a natural substance isolated from cultures of thefungus Cercosporidium heningsii (see U.S. Pat. No. 4,983,587, TheJournal of Organic Chemistry, 1991, Vol. 56, pp. 909-910, Tetrahedron,1992, Vol. 48, pp. 4757-4766, and Molecular and Cellular Biology, 1994,Vol. 14, pp. 1017-1025).

Cercosporamide is known to have antifungal activity. In addition, somederivatives of cercosporamide are also known to have antifungal activitysimilar to that of cercosporamide (U.S. Pat. No. 4,983,587).

Cercosporamide is also useful as a precursor for synthesizingderivatives thereof, and there is currently a need for a more efficientproduction process.

Although the fungus Cercosporidium heningsii is known to be amicroorganism that produces cercosporamide, fungi belonging to the genusLachnum and Pseudaegerita were not known to produce cercosporamide.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a cercosporamideproduction process using microorganisms. Another object of the presentinvention is to provide cercosporamide produced according to thatproduction process. Still another object of the present invention is toprovide a microorganism that produces cercosporamide.

As a result of conducting extensive studies to solve the aforementionedproblems, the inventors of the present invention found thatcercosporamide is produced by fungi belonging to the genera Lachnum andPseudaegerita, and also discovered a process for producingcercosporamide by using those fungi, thereby leading to completion ofthe present invention.

Means for Solving the Problems

The present invention is composed of the following:

(1) a process for producing cercosporamide comprising: culturing afungus belonging to the genus Lachnum and yielding cercosporamide fromthe culture thereof,

(2) the process for producing cercosporamide described in (1) above,wherein the fungus belonging to the genus Lachnum is at least one fungusselected from the group consisting of Lachnum fuscescens, Lachnumcalycioides and Lachnum caesaliatum,

(3) the process for producing cercosporamide described in (1) above,wherein the fungus belonging to the genus Lachnum is at least one fungusselected from the group consisting of Lachnum fuscescens SANK 19096,Lachnum calycioides SANK 12497 and Lachnum caesaliatum SANK 10906,(4) a process for producing cercosporamide comprising: culturing afungus belonging to the genus Pseudaegerita, and yielding cercosporamidefrom the culture thereof,(5) the process for producing cercosporamide described in (4) above,wherein the fungus belonging to the genus Pseudaegerita is Pseudaegeritawebsteri,(6) the process for producing cercosporamide described in (4) above,wherein the fungus belonging to the genus Pseudaegerita is Pseudaegeritawebsteri SANK 11006,(7) cercosporamide produced according to the production processdescribed in any one of (1) to (6) above,(8) a pharmaceutical containing as an active ingredient thereofcercosporamide produced according to the production process described inany one of (1) to (6) above,(9) Lachnum fuscescens SANK 19096,(10) Lachnum calycioides SANK 12497,(11) Lachnum caesaliatum SANK 10906, and,(12) Pseudaegerita websteri SANK 11006.

In the present specification, “cercosporamide” refers to the compoundrepresented by the following formula (1).

Cercosporamide exhibits the physicochemical properties indicated below.

-   1) Appearance: Weakly acidic, liposoluble, yellowish-brown powder-   2) Molecular formula: C₁₆H₃NO₇-   3) Molecular weight: 331 (as determined by EI mass spectrometry)-   4) The exact mass as observed by high-resolution EI mass    spectrometry [M]⁺ (as C₁₆H₁₃NO₇) is as indicated below.

Found: 331.0690

Calculated: 331.0692

-   5) Specific rotatory power: [α]_(D) ²⁵ −396.4° (c 0.51,    acetonitrile)-   6) Proton-Nuclear magnetic resonance spectrum: (δ, ppm)

The ¹H-nuclear magnetic resonance spectrum (500 MHz) as measured indeuterated heavy chloroform using the residual proton signal (7.27 ppm)as an internal standard is as indicated below.

1.75 (3H, s), 2.68 (3H, s), 5.99 (1H, s), 6.26 (1H, s), 6.38 (1H, s),6.96 (1H, s), 10.55 (1H, s), 12.98 (1H, s), 18.83 (1H, s)

-   7) Carbon-13-Nuclear magnetic resonance spectrum: (δ, ppm)

The ¹³C-nuclear magnetic resonance spectrum (125 MHz) as measured indeuterated chloroform using the deuterated chloroform signal (77.23 ppm)as an internal standard is as indicated below.

27.9 (q), 32.1 (q), 58.8 (s), 94.0 (s), 99.4 (d), 101.5 (d), 104.0 (s),105.6 (s), 155.2 (s), 158.1 (s), 166.1 (s), 169.8 (s), 178.2 (s), 191.8(s), 197.7 (s), 201.9 (s)

Effects of the Invention

According to the present invention, a process for producingcercosporamide, cercosporamide produced according to said productionprocess, and a fungus belonging to the genus Lachnum or the genusPseudaegerita that produces cercosporamide are provided. Specificexamples of fungi provided by the present invention that producecercosporamide include Lachnum fuscescens SANK 19096, Lachnumcalycioides SANK 12497, Lachnum caesaliatum SANK 10906 and Pseudaegeritawebsteri SANK 11006. In addition to cercosporamide itself being usefulas an antifungal agent, it is also useful as a precursor for producingderivatives of cercosporamide.

BEST MODE FOR CARRYING OUT THE INVENTION 1. Cercosporamide-ProducingFungi

As a result of conducting an extensive search for microorganisms thatproduce cercosporamide, the inventors of the present invention foundcercosporamide in the cultures of Lachnum fuscescens SANK 19096, Lachnumcalycioides SANK 12497, Lachnum caesaliatum SANK 10906 and Pseudaegeritawebsteri SANK 11006.

The fungus that produces cercosporamide is not particularly limited solong as it belongs to the genus Lachnum and Pseudaegerita, it ispreferably at least one fungus selected from the group consisting ofLachnum fuscescens, Lachnum calycioides, Lachnum caesaliatum andPseudaegerita websteri, and more preferably at least one fungus selectedfrom the group consisting of Lachnum fuscescens SANK 19096 (hereinafterreferred to as “strain SANK 19096”), Lachnum calycioides SANK 12497(hereinafter referred to as “strain SANK 12497”), Lachnum caesaliatumSANK 10906 (hereinafter referred to as “strain SANK 10906”) andPseudaegerita websteri SANK 11006 (hereinafter referred to as “strainSANK 11006”).

The following indicates the mycological properties of strain SANK 19096,strain SANK 12497, strain SANK 10906 and strain SANK 11006.

In describing mycological characteristics, indication of colony coloringwas in accordance with “Kornerup, A. and Wanscher, J. H. (1978), MethuenHandbook of Colour, 3rd ed., Eyre Methuen, London, UK, 1-252”.

Observation of mycological properties during culturing of each fungusused the media indicated below.

Potato dextrose agar medium (hereinafter referred to as “PDA medium”):39 g of Nissui Potato Dextrose Agar Medium (Nissui Pharmaceutical Co.,Ltd.) is dissolved in 1000 ml of distilled water followed by sterilizingfor 15 minutes at 121° C. and preparing plates.

Modified Weitzman and Silva-Hutner medium (hereinafter referred to as“WSH medium”): 10 g of Nisshoku Oatmeal, 1 g of magnesium sulfateheptahydrate, 1 g of potassium dihydrogenphosphate, 1 g of sodiumnitrate and 20 g of agar are dissolved in 1000 ml of distilled waterfollowed by sterilizing for 15 minutes at 121° C. and preparing plates.

Malt extract agar medium (hereinafter referred to as “MEA medium”): 20 gof malt extract, 1 g of polypepton (Nihon Pharmaceutical Co., Ltd.), 20g of glucose and 20 g of agar are dissolved in 1000 ml of distilledwater followed by sterilizing for 15 minutes at 121° C. and preparingplates.

Corn meal agar medium (hereinafter referred to as “CMA medium”): 17 g ofNissui Corn Meal Agar (Nissui Pharmaceutical Co., Ltd.) is dissolved in1000 ml of distilled water followed by sterilizing for 15 minutes at121° C. and preparing plates.

Abdullah's malt extract agar medium (hereinafter referred to as “AMEAmedium”): 0.76 g of malt extract, 0.14 g of glycerol, 0.16 g of dextrin,0.046 g of GE90M (DMV Co., Ltd.) and 15 g of agar are dissolved in 1000ml of distilled water followed by sterilizing for 15 minutes at 121° C.and preparing plates.

PDA medium, WSH medium, CMA medium and MEA medium were used for strainSANK 19096, strain SANK 12497 and strain SANK 10906, while PDA medium,WSH medium, CMA medium and AMEA medium were used for strain SANK 11006.

(1) Lachnum fuscescens SANK 19096

Lachnum fuscescens SANK 19096 was isolated as Lachnum sp. SANK 19096from fruiting bodies formed on fallen leaf collected in NaganoPrefecture, Japan.

The mycological properties of a dried fruiting body specimen used toisolate strain SANK 19096 are described as follows. Apothecium is shortstipe; the disc is slightly concaved, 557 μm in diameter and pale brownwhen rehydrated with distilled water. The disc is deep cupulate withincurving margin, 499 μm in diameter and pale brown when dry. The ectalexcipulum is “textura prismatica”, hyaline to subhyaline, composed ofthick-walled cells and with short hair-like projections or hairs fromthe outermost layer. The medullary excipulum is “textura intricata” andhyaline. The hairs are cylindrical, straight or slightly curved, obtuseat the apex, multi-septate, granulate all over, brown, 4.5 to 6.0 μm inwidth and 71 to 76 μm in length. Amorphous resinous matters are rarelyattached to the ends of the hairs. The stipe is cylindrical, pale brownand 115 μm in height. The asci are inoperculate, cylindrical-clavate,8-spored, 30 to 43 μm in length and 3.5 to 5 μm in width. The tip of theascus is stained blue by Melzer's reagent. The paraphyses arelanceolate, aseptate or occasionally 1- or 2-septate, hyaline, 50.9 to68.7 μm in length, 2.7 to 4.1 μm at the widest point and exceed the asciby 9.1 to 16.5 μm. The ascospores are fusiform to oblong-ellipsoid,aseptate, hyaline, 5.5 to 8.5 in length and 1.3 to 2.2 μm in width.

Strain SANK 19096 demonstrates the mycological properties indicatedbelow on the various media described above.

Colonies on PDA medium reach a diameter of 19 to 21 mm followingculturing for 21 days at 23° C. The colonies are velvety to cottony,composed of a mycelia layer protruding toward the center and reddishgray (11B2) to white. Exudates, sclerotia and conidia are not observed.The back of the colonies is grayish orange (5B4) in the center and whitealong the margins, have radiating wrinkles. Soluble pigment is notobserved. Mycelia are septate, branched, hyaline and 1.5 to 3.5 μm indiameter.

Colonies on WSH medium reach a diameter of 29 to 31 mm followingculturing for 21 days at 23° C. The colonies are floccose to velvety,composed of a thin mycelia layer and white. Exudates, sclerotia andconidia are not observed. The back of the colonies is white. Solublepigments are not observed.

Colonies on CMA medium reach a diameter of 33 to 34 mm followingculturing for 21 days at 23° C. The colonies consist only of anextremely thin mycelia layer and are colorless. Exudates, sclerotia andconidia are not observed. The back of the colonies is colorless. Solublepigments are not observed.

Colonies on MEA medium reach a diameter of 21 to 22 mm followingculturing for 21 days at 23° C. The colonies are cottony in the center,mycelia concentrate toward the margins and become velvety and grayishyellow (4C4) to yellowish white (4A1), while the concentrated portion ofairborne mycelia are white. The margins are composed only of basalmycelia and brownish orange (5C3) to orange gray (5C2). Exudates,sclerotia and conidia are not observed. The back of the colonies isyellowish brown (5D6) to grayish orange (5B3) and white along themargins. Soluble pigments are not observed.

The morphological characteristics of the present fungus agreed well withthe description of Lachnum species in the document by Spooner (Spooner,B M (1987) Bibliotheca Mycologica 116: 1-711). Thus, this fungus wasidentified as Lachnum sp. and named Lachnum sp. strain SANK 19096.

Strain SANK 19096 agreed, with only few exceptions, with thedescriptions of Dasyscyphus fuscescens (Pers.) Gray of the document byDennis, R W G (Dennis, R W G (1949) Mycological Papers 32: 1-97) andLachnum fuscescens (Pers.) P. Karst. in the document by Tanaka andHosoya (Tanaka, I and Hosoya, T (2001) Mycoscience 42: 597-609).Dasyscyphus fuscescens is currently treated as a synonym of Lachnumfuscescens. In addition, in a document by Baral and Krieglsteiner(Baral, H O and Krieglsteiner, G J (1985) Beihefte zur Zeitschrift fürMykologie 6: 1-160), a new genus Brunnipila is established for Lachnumspecies having brown hairs in the manner of Lachnum fuscescens, and istreated as a different genus. In the document by Baral andKrieglsteiner, Lachnum fuscescens is treated as a synonym of Brunnipilafuscescens (Pers.) Baral. However, this approach is not widely accepted,and in the publication by Krik, et al. (Krik, P M et al. (2001)Ainsworth & Bisby's Dictionary of Fungi 9th Edition, CABI International,Wallingford, UK, 1-655), the genus Brunnipila is treated as a synonym ofthe genus Lachnum. Thus, in the present patent, strain SANK 19096 wasidentified as Lachnum fuscescens in accordance with the document byTanaka and Hosoya, and was named Lachnum fuscescens SANK 19096.

Strain SANK 19096 was internationally deposited as Lachnum sp. SANK19096 on May 24, 2005 at the International Patent Organism Depository ofthe National Institute of Advanced Industrial Science and Technology(location: Chuo #6, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture,Japan), and was assigned the deposit number FERM BP-10338.

(2) Lachnum calycioides SANK 12497

The mycological properties of strain SANK 12497 are indicated below.

Lachnum calycioides SANK 12497 was isolated from fruiting bodies formedon the dried stems of grass collected in Tottori Prefecture, Japan.

The mycological properties of dried fruiting body specimens used toisolate strain SANK 12497 are described as follows. Apothecia arescattered or clustered, stipitate, the margins curve to the inside whendry, 1271 to 1817 μm in height and 437 to 913 μm in diameter; thesurface are grayish orange (6B4); the hairs are light brown (7D4) todark brown (7F7); the stipes and ectal excipulum are grayish orange(6B4). The ectal excipulum is “textura prismatica”, hyaline to brown incolor, composed of thick-walled cells and with short hair-likeprojections or hairs from the outermost layer. The medullary excipulumis “textura intricata” and hyaline. The hairs are cylindrical, straightor somewhat curved, obtuse at the apex, multi-septate, granulate allover, brown, 3.8 to 5.5 μm in width and 58.1 to 111.6 μm in length.Amorphous matters are attached to the ends of the hairs, and ascosporesare frequently attached thereon. The stipes are cylindrical, 908 to 1374μm in length, 170 to 205 μm in diameter and hyaline to brown. The asciare inoperculate, cylindrical-clavate, conical apex, 8-spored, 69.3 to80.2 μm in length and 3.9 to 6.8 μm in width. The tip of the ascus isstained blue by Melzer's reagent following treatment with 3% KOH. Theparaphyses are lanceolate, aseptate or occasionally 1- or 3-septate,hyaline, 98.1 to 130.2 in length, 4.4 to 7.0 μm at the widest point andexceed the asci by 13.5 to 36.0 μm. The ascospores are fusiform tooblong-ellipsoid, aseptate, hyaline, 8.8 to 15.6 μm in length and 1.8 to3.0 μm in width.

Strain SANK 12497 demonstrates the mycological properties indicatedbelow on the various media described above.

Colonies on PDA medium reach a diameter of 21 to 25 mm followingculturing for 21 days at 23° C. The colonies are velvety to cottony,composed of a mycelia layer somewhat protruding toward the center andreddish brown (8E2) to grayish orange (5B3). The margins of the coloniesare irregularly laciniated. Exudates, sclerotia and conidia are notobserved. The back of the colonies is dark brown (6F4) to grayish orange(5B3) in the center and white along the margins, having radiatingwrinkles. Soluble pigment is not produced. Mycelia are septate,branched, hyaline to pale brown and 1.3 to 2.6 μm in diameter.

Colonies on WSH medium reach a diameter of 32 to 34 mm followingculturing for 21 days at 23° C. The colonies are composed of a thinmycelia layer, light yellow (2A5) to white. The margins of the coloniesare smooth. Exudates, sclerotia and conidia are not observed. The backof the colonies is the same as that of the surface. Soluble pigment isnot produced.

Colonies on CMA medium reach a diameter of 33 to 35 mm followingculturing for 21 days at 23° C. The colonies are composed only of anextremely thin mycelia layer and colorless. The margins of the coloniesare smooth. Exudates, sclerotia and conidia are not observed. The backof the colonies has the same color as that of the surface. Solublepigment is not produced.

Colonies on MEA medium reach a diameter of 18 to 27 mm followingculturing for 21 days at 23° C. The colonies are thin and velvety, havewrinkles radiating toward the margins and are grayish orange (5B6) tolight yellow (3A4). The margins of the colonies are rarely gentlyinvoluted. Exudates, sclerotia and conidia are not observed. The back ofthe colonies is grayish orange (5B6) to orange gray (5B2), and whitealong the margins. Soluble pigment is not produced.

The morphological characteristics of the present fungus agreed well withthe description of Lachnum species in the document by Spooner (Spooner,B M (1987) Bibliotheca Mycologica 116: 1-711). Moreover, morphologicalcharacteristics also agreed, with only few exceptions, with thedescriptions of Lachnum calycioides (Rehm) Rehm of the publication byRehm (Rehm, H (1986) Dr. Rabenhorst's Kryptogamen-Flora von Deutschland,Oesterreichs und der Schweiz Zweite Auflage. Vol. 1. III. Abtheilung:Ascomyceten: Hysteriaceen und Discomyceten, Eduard Kummer, Leipzig,Germany, 1-1275), Dasyscyphus calycioides (Rehm) Sacc. of thepublication by Brietenbach and Kranzlin (Brietenbach, J & Kranzlin, F(1984) Fungi of Switzerland. Vol. 1 Ascomycetes, Verlag Mykologia,Luzern Switzerland, 1-310), and Brunnipila calycioides (Rehm) Baral ofthe document by Scheuer (Scheuer, C (1988) Bibliotheca Mycologica 123:1-274). Lachnum calycioides, Dasyscyphus calycioides and Brunnipilacalycioides are synonyms of the same species. Dasyscyphus calycioides isconsidered to be a synonym of Brunnipila calycioides in the document byBaral and Krieglsteiner (Baral, H O and Krieglsteiner, G J (1985)Beihefte zur Zeitschrift für Mykologie 6: 1-160). However, the genusBrunnipila used in the document of Scheuer (1988) is not widelyaccepted, and the genus Brunnipila is treated as a synonym of the genusLachnum in the publication by Krik, et al. (Krik, P M et al. (2001)Ainsworth & Bisby's Dictionary of Fungi 9th Edition, CABI International,Wallingford, UK, 1-655). Thus, in the present patent, strain SANK 12497was identified as Lachnum calycioides and named Lachnum calycioides SANK12497.

Strain SANK 12497 was internationally deposited as Lachnum calycioidesSANK 12497 on Jun. 29, 2006 at the International Patent OrganismDepository of the National Institute of Advanced Industrial Science andTechnology (location: Chuo #6, 1-1-1 Higashi, Tsukuba City, IbarakiPrefecture, Japan), and was assigned the deposit number FERM BP-10636.

(3) Lachnum caesaliatum SANK 10906

The mycological properties of strain SANK 10906 are indicated below.

Lachnum caesaliatum SANK 10906 was isolated from fruiting bodies formedon fallen leaves collected in Kanagawa Prefecture, Japan.

The mycological properties of dried fruiting body specimens used toisolate strain SANK 10906 are described as follows. Apothecia arescattered or clustered, stipitate, the margins curve to the inside whendry, dark orange (5A8), 391 to 858 μm in height and 212 to 446 μm indiameter; the hairs are white; the stipes and ectal excipulum are lightorange (6A5). The discs are 532 to 739 μm, slightly concave and paleorange color overall when rehydrated with distilled water.

The ectal excipulum is “textura prismatica”, hyaline to pale brown,composed of thick-walled cells and with short hair-like projections orhairs from the outermost layer. The medullary excipulum is “texturaintricata” and hyaline. The hairs are cylindrical, straight or slightlycurved, obtuse at the apex, multi-septate, granulate all over, hyaline,2.1 to 2.7 μm in width and 43.9 to 98.9 μm in length. The stipes arecylindrical, hyaline to pale brown, 310 to 530 μm in length and 55 to 90μm in diameter. The asci are inoperculate, cylindrical-clavate,8-spored, 29.8 to 42.0 μm in length and 3.6 to 5.4 μm in width. The tipof the ascus is stained blue by Melzer's reagent following treatmentwith 3% KOH. The paraphyses are lanceolate, aseptate, hyaline, 33.9 to42.6 μm in length, 1.4 to 2.8 μm at the widest point and exceed the asciby 5 to 9.5 μm. The ascospores are fusiform to oblong-ellipsoid,aseptate, hyaline, 11.4 to 17.2 μm in length and 1.5 to 2.4 μm in width.

Strain SANK 10906 demonstrates the mycological properties indicatedbelow on the various media described above.

Colonies on PDA medium reach a diameter of 10 to 12 mm followingculturing for 21 days at 23° C. The colonies are velvety to cottony andgrayish yellow (4C7) to light yellow (4A5). The margins of the coloniesare white and irregularly laciniated. Exudates, sclerotia and conidiaare not observed. The back of the colonies is yellowish brown (5F8-5E6)in the center and white along the margins. Soluble pigment is notproduced. Mycelia are septate, branched, light brown to hyaline and 1.7to 5.1 μm in diameter.

Colonies on WSH medium reach a diameter of 17 to 20 mm followingculturing for 21 days at 23° C. The colonies are composed of a thinmycelia layer, partially form white, cottony, airborne mycelia and arewhite. The margins of the colonies are smooth. Exudates, sclerotia andconidia are not observed. The back of the colonies is white. Solublepigments are not produced.

Colonies on CMA medium reach a diameter of 30 to 34 mm followingculturing for 21 days at 23° C. The colonies consist only of anextremely thin mycelia layer and are colorless. The margins of thecolonies are smooth. Exudates, sclerotia and conidia are not observed.The back of the colonies is colorless. Soluble pigments are notproduced.

Colonies on MEA medium reach a diameter of 8 to 11 mm followingculturing for 21 days at 23° C. The colonies are velvety and brownishorange (5C4) to grayish orange (5B3). The margins of the colonies arewhite and rarely gently laciniated. Exudates, sclerotia and conidia arenot observed. The back of the colonies is yellowish brown (5F8) togolden brown (5D8) and white along the margins. Soluble pigments are notproduced.

The morphological characteristics of the present fungus agreed well withthe description of Lachnum species in the document by Spooner (Spooner,B M (1987) Bibliotheca Mycologica 116: 1-711). Among existing Lachnumspecies, strain SANK 10906 resembles Lachnum pteridophyllum (Rodway)Spooner and Lachnum varians (Rehm) M. P. Sharma described in thedocument by Spooner. However, in contrast to strain SANK 10906 beingisolated from fallen leaves of woody plants, Lachnum pteridophyllum andLachnum varians are clearly distinguished from strain SANK 10906 sincethey previously have only been isolated from Cyatheaceae hosts and havelonger asci and broader hairs than strain SANK 10906. Therefore, strainSANK 10906 was identified as a new species of the genus Lachnum and istentatively named as Lachnum caesaliatum, and subsequently named Lachnumcaesaliatum SANK 10906.

Strain SANK 10906 was internationally deposited as Lachnum caesaliatumSANK 10906 on Jun. 29, 2006 at the International Patent OrganismDepository of the National Institute of Advanced Industrial Science andTechnology (location: Chuo #6, 1-1-1 Higashi, Tsukuba City, IbarakiPrefecture, Japan), and was assigned the deposit number FERM BP-10634.

(4) Pseudaegerita websteri SANK 11006

The mycological properties of strain SANK 11006 are indicated below.

Pseudaegerita websteri SANK 11006 was isolated from propagules formed onfallen branches collected in Miyazaki Prefecture, Japan.

The mycological properties of propagules on AMEA medium followingculturing for 28 days at 15° C. and on dried specimens used forisolation of strain SANK 11006 are described below.

Mycelia on AMEA medium are superficial or immersed, septate, branched,brown to hyaline and 1.3 to 3.2 μm in diameter. Conidiophores aresolitary, micronematous or semi-macronematous and septate. Conidiogenouscells are polyblastic. Propagules are globose to subglobose, scatteredon the surface of the substrate, rarely aggregated, white and 26.6 to80.9 μm in diameter. Propagules are composed of clusters of highlybranched structures, and each cell is spherical to ellipsoidal and 3.6to 5.6 μm in diameter. There are spaces between each cell, eachsuccessively budding out 1 to 4 daughter cells. Phialidic conidia arenot observed. Propagules formed on fallen branches are nearly identicalto those on AMEA medium, 37.0 to 106.0 μm in diameter, and each cell is3.4 to 6.8 μm in diameter. Phialidic conidia are not observed.

Strain SANK 11006 demonstrates the mycological properties indicatedbelow on the various media described above.

Colonies on PDA medium reach a diameter of 13 to 14 mm followingculturing for 28 days at 15° C. The colonies are velvety, composed of amycelia layer protruding toward the center and olive (2F6) to olive gray(2D2). The margins of the colonies are slightly laciniated. Exudates,sclerotia and propagules are not observed. The back of the colonies isthe same color as the surface. Soluble pigment is not produced. Myceliaare septate, branched, reddish brown to hyaline and 1.6 to 3.0 μm indiameter.

Colonies on WSH medium reach a diameter of 13 mm following culturing for28 days at 15° C. The colonies are composed of a thin mycelia layer andwooly airborne mycelia and are olive (2F4-2E5). The margins of thecolonies are smooth. Exudates, sclerotia and propagules are notobserved. The back of the colonies exhibits olive (2F7) to olive gray(2F2). Soluble pigments are not produced.

Colonies on CMA medium reach a diameter of 13 to 18 mm followingculturing for 28 days at 15° C. The colonies consist only of a thinmycelia layer and are olive (3F3) to grayish yellow (3B3). The marginsof the colonies are smooth. White propagules are sparsely produced fromthe center to the margins of the colonies. Exudates and sclerotia arenot observed. The back of the colonies is the same color as the surface.Soluble pigments are not produced.

Colonies on AMEA medium reach a diameter of 6 to 16 mm followingculturing for 28 days at 15° C. The colonies are composed only of a thinmycelia layer and are olive (3F3) to grayish yellow (3B3). Whitepropagules are sparsely produced from the center to the margins of thecolonies. Exudates and sclerotia are not observed. The back of thecolonies is the same color as the surface. Soluble pigments are notproduced.

The morphological characteristics of the present fungus agreed, withonly few exceptions, with the description of species of Pseudaegeritawebsteri Abdullah & Guarro of the document by Abdullah et al. (Abdullah,S K et al. (2005) Mycological Research 109: 590-594). There were onlysmall differences between strain SANK 11006 and the descriptions ofPseudaegerita websteri, with the diameter of individual cells of thepropagules being described as 3 to 4 μm for Pseudaegerita websteri andbeing described as 3.6 to 5.6 μm for strain SANK 11006. Thus, thisfungus was identified as Pseudaegerita websteri, and named Pseudaegeritawebsteri SANK 11006.

Strain SANK 11006 was internationally deposited as Pseudaegeritawebsteri SANK 11006 on Jun. 29, 2006 at the International PatentOrganism Depository of the National Institute of Advanced IndustrialScience and Technology (location: Chuo #6, 1-1-1 Higashi, Tsukuba City,Ibaraki Prefecture, Japan), and was assigned the deposit number FERMBP-10635.

As is commonly known, fungi are susceptible to mutation either naturallyor by artificial manipulation (such as by irradiating with ultravioletlight, irradiating with radiation or treating with chemicals or drugs),and this applies similarly to the strains SANK 19096, SANK 12497, SANK10906 and SANK 11006 of the present invention. All mutant strains ofthese strains SANK 19096, SANK 12497, SANK 10906 and SANK 11006 areincluded in the present invention.

In addition, strains obtained by genetic techniques such asrecombination, transduction or transformation are also included in thesemutant strains. Namely, cercosporamide-producing strain SANK 19096,strain SANK 12497, strain SANK 10906, strain SANK 11006, mutant strainsthereof and mycotic strains not clearly distinguished therefrom are allincluded in strains SANK 19096, SANK 12497, SANK 10906 and SANK 11006.

2. Fermentation

Cercosporamide producers can be cultured using media containing a carbonsource, a nitrogen source and an inorganic salt capable of beingassimilated by microorganisms in the manner of that ordinarily used forfermentative production by microorganisms.

Examples of carbon sources include glucose, fructose, maltose, sucrose,mannitol, glycerin, dextrin, oats, rye, pressed barley, cornstarch,potato, cornmeal, soybean meal, cottonseed oil, molasses, citric acidand tartaric acid, and these can be used alone or two or more can beused in combination. The content of the carbon source normally variesbetween 1 and 10% by weight of the amount of medium.

Examples of nitrogen sources that can be used include substancescontaining protein or hydrolysates thereof and inorganic salts. Specificexamples of such nitrogen sources include soybean meal, wheat bran,peanut meal, cottonseed oil, casein hydrolysates, farmamine, fish meal,corn steep liquor, peptone, meat extract, yeast, yeast extract, maltextract, sodium nitrate, ammonium nitrate and ammonium sulfate, andthese can be used alone or two or more can be used in combination. Thecontent of the nitrogen source normally varies between 0.1 and 10% byweight of the amount of medium.

Examples of nutritive inorganic salts that can be used include ordinarysalts and metal salts capable of providing ions. Specific examples ofsalts include sodium, ammonium, calcium, phosphate, sulfate, chlorideand carbonate salts. Specific examples of metal salts include potassium,calcium, cobalt, manganese, iron and magnesium salts.

In addition, vitamins such as vitamin B1 or biotin, mycelium growthpromoters such as thiamine and the like may also be added if necessary.

For liquid culturing of cercosporamide producers, silicone oil,vegetable oil or surfactant and the like can be used as an antifoamingagent. Although the pH of the medium when culturing any fungus selectedfrom the group consisting of strain SANK 19096, strain SANK 12497,strain SANK 10906 and strain SANK 11006 for the purpose of production ofcercosporamide varies according to the medium components, growthtemperature and the like, a pH ordinarily used for the production offermentation products may be adopted.

Although varying according to medium components, pH and the like, thetemperature for the fermentation of strain SANK 19096, strain SANK12497, strain SANK 10906 and strain SANK 11006 is normally 10 to 30° C.,and a temperature range of 20 to 26° C. is favorable for growth. Afermentation temperature of 23 to 26° C. is preferred for the productionof cercosporamide.

There are no particular limitations on the fermentation method forproducing cercosporamide, and any method commonly used to culturemicroorganisms may be used. Examples of methods that can be used includesolid culture, agitation culture, shaking culture and aeration-agitationculture.

Although the following indicates a fermentation method of culturingstrain SANK 19096 as an example of a method for producingcercosporamide, the fermentation method is not limited to this methodprovided it is used to produce cercosporamide.

Fermentation of strain SANK 19096 normally starts with a seed culturecomprised of one or two or more stages followed by shaking on a rotaryshaker for 5 to 10 days (and preferably 7 days) at 20 to 26° C. (andpreferably 26° C.) or until the seed culture grows sufficiently.

In the case of solid culturing of strain SANK 19096, the aforementionedseed culture broth can be inoculated into a final fermentation medium ina solid medium culture vessel such as an Erlenmeyer flask. Fermentationis carried out in the form of a standing culture by allowing the solidculture vessel to stand for 10 to 20 days (and preferably 14 days) at 20to 26° C. (and preferably 26° C.). The desired cercosporamide can beobtained from final fermentation medium (cultured material).

In the case of liquid culturing of strain SANK 19096 on a comparativelysmall scale, the seed culture broth is inoculated into an Erlenmeyerflask containing the final fermentation medium followed by shaking on arotary shaker at 20 to 26° C. (and preferably 26° C.) for several days.The desired cercosporamide can be obtained from the resulting fermentedbroth (culture).

On the other hand, in the case of liquid culturing of strain SANK 19096on a comparatively large scale, it is preferred to use a suitablefermentation tank equipped with an agitator, an aeration apparatus,temperature controller and the like. The use of such an apparatus makesit possible to prepare the medium in advance in said tank. For example,the final production medium is heated at 121° C. for sterilization andthen chilled. Next, the final production medium is inoculated with theseed culture followed by final fermentation while aerating and agitatingat 26° C. The desired cercosporamide can be obtained from the resultingfermented broth (cultured material). This type of fermentation methodallows the desired cercosporamide to be produced in large quantities.

In the case of producing cercosporamide by fermentation of acercosporamide producer of the present invention, the amount ofcercosporamide produced in said culture can be monitored at timeintervals by high-performance liquid chromatography (HPLC) and the liketo be described later. In the case of monitoring by HPLC analysis, theproduct is previously extracted from the culture with a hydrophilicsolvent such as acetone followed by evaporation of said hydrophilicsolvent and extracting with a solvent that is not miscible with watersuch as ethyl acetate. Next, after evaporation of the water-immisciblesolvent, the product dissolved in a suitable solvent is preferablysubjected to analysis or testing.

3. Extraction and Purification of Cercosporamide

Cercosporamide can be extracted from a fermented solid medium (culturedmaterial) or a fermented liquid medium (cultured material) of acercosporamide producer according to a method indicated in the previoussection “2. Fermentation”. There are no particular limitations on themethods used to extract and purify cercosporamide providingcercosporamide can be extracted and purified.

For example, after the completion of liquid fermentation of acercosporamide producer, an organic solvent miscible with water such asacetone is added to the fermented broth (cultured material), the extractis separated into a soluble fraction (culture supernatant) and insolublefraction (mycelium) by a filtration procedure using diatomaceous earthas an aid, or by a centrifugation procedure, and cercosporamide presentin the resulting culture supernatant can be further extracted andpurified by utilizing the physicochemical properties of thecercosporamide. In addition, as another method, after adding afiltration aid such as Celite to a fermented broth (cultured material)following completion of liquid fermentation of a cercosporamideproducer, the fermented broth is filtered, the insoluble fractioncontaining the resulting mycelium is immersed in an aqueous solution ofa solvent miscible with water such as acetone, and the cercosporamidecan then be yielded by further extraction and purification from themycelium extract by utilizing the physicochemical properties thereof.

Examples of methods for the purification of cercosporamide from culturesupernatant include, but are not limited to, an extraction method byadding an acidic substance such as hydrochloric acid to the culturesupernatant to adjust the pH to 3.0 followed by the addition of ethylacetate.

The resulting extract can be purified by adsorption columnchromatography using a carrier such as silica gel or magnesium-silicagel-based Florisil, thin layer chromatography, partition columnchromatography using TSK Gel ToyoPearl HW-40F (registered trademark,Tosoh Corp.) or Sephadex LH-20 (registered trademark, AmershamBiosciences K.K.) and the like, reverse phase column chromatographyusing Cosmosil 140C18 (registered trademark, Nacalai Tesque Inc.), orHPLC using a normal phase or reversed phase column to isolate thedesired cercosporamide.

The behavior of the compound in each purification step duringpurification of the cercosporamide of the present invention can bemonitored, for example, by HPLC under the conditions indicated below.

(HPLC Conditions for Detecting Cercosporamide)

Separation column: YMC J'sphere ODS-H80 S-4, 4.6×150 mm (YMC Co., Ltd.)

Mobile phase: acetonitrile: 0.4% triethylamine-phosphate buffer (pH 3.2)(45:55)

Flow rate: 1.0 ml/minute

Detection: Ultraviolet absorption at, 225 nm

Retention time: 9.3 minute

4. Pharmaceutical Medicine Containing Cercosporamide

Cercosporamide of the present invention, or a salt thereof, can be invarious forms when administered as a pharmaceutical medicine to a humanor to an animal other than a human. The administration form thereof isdependent on the preparation, age, gender, disease and the like. Forexample, a tablet, pill, powder, granule, syrup, liquid, suspension,emulsion, granule, capsule or the like is administered orally. Aninjection preparation or the like is administered by intravenousadministration, intramuscular administration, intracutaneousadministration, subcutaneous administration or intraperitonealadministration. A suppository is administered rectally. An ointment orthe like is used as an externally applied preparation.

A pharmaceutical preparation containing cercosporamide as an activeingredient thereof can be produced in accordance with ordinary methodsusing known auxiliaries normally able to be used in the field ofpharmaceutical preparations, examples of which include excipients,binders, disintegrants, lubricants, dissolving agents, corrigents andcoatings.

When forming into the form of a tablet, carriers known in the relevantfield can be widely used for the carrier, examples of which includeexcipients such as lactose, saccharose, sodium chloride, glucose, urea,starch, calcium carbonate, kaolin, crystalline cellulose or silicicacid, binders such as water, ethanol, propanol, simple syrup, glucosesolution, starch solution, gelatin solution, carboxymethyl cellulose,shellac, methyl cellulose, potassium phosphate or polyvinyl pyrrolidone,disintegrants such as dry starch, sodium alginate, powdered agar,powdered laminarin, sodium hydrogencarbonate, calcium carbonate,polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate,monoglyceride stearate, starch or lactose, disintegration inhibitorssuch as saccharose, stearine, cocoa butter or hydrogenated oil,absorption promoters such as quaternary ammonium salts or sodium laurylsulfate, moisture retention agents such as glycerin or starch,adsorbents such as starch, lactose, kaolin, bentonite or colloidalsilicic acid, and lubricants such as purified talc, stearates, powderedboric acid or polyethylene glycol. Tablets can be provided with anordinary coating as necessary, such as sugar-coated tablets,gelatin-coated tablets, enteric-coated tablets, film-coated tablets ordouble-layer or multilayer tablets.

When forming into the form of a pill, carriers known in the relevantfield can be widely used for the carrier, examples of which includeexcipients such as glucose, lactose, starch, cocoa butter, hydrogenatedoil, kaolin or talc, binders such as powdered gum Arabic, powderedtragacanth, gelatin or ethanol, and disintegrants such as laminarin oragar.

In the case of formulating as an injection preparation, it is preferablethat a liquid or suspension be sterilized and be made isotonic withblood, and when forming into the form of a liquid, emulsion orsuspension, diluents known in the relevant field can be widely used forthe diluent, examples of which include water, ethyl alcohol, propyleneglycol, epoxidated isostearyl alcohol, epoxidated stearyl alcohol,polyoxidated isostearyl alcohol and polyoxyethylene sorbitan fatty acidesters. An adequate amount of salt, glucose or glycerin may be containedto maintain isotonicity. Dissolving aids, buffers, indolent sugars,colorants, preservatives, fragrances, flavorings, sweeteners or otherdrugs and the like may also be contained.

Furthermore, in the case of intravenous administration of an injectionpreparation, the injection preparation is administered alone, aftermixing with an ordinary supplement such as glucose or amino acid, or asan emulsion with a polyoxyethylene sorbitan fatty acid ester and thelike.

When forming into the form of a suppository, carriers known in therelevant field can be widely used for the carrier, examples of whichinclude polyethylene glycol, cocoa butter, higher alcohols, esters ofhigher alcohols, gelatin and semi-synthetic glycerides.

When forming into the form of an externally applied preparation such asointment, an excipient known in the relevant field belonging to any of ahydrophobic base (oily ointment base), hydroscopic base, hydrophilicbase (cream) and water-soluble base (non-grease-based ointment) can bewidely used for the excipient.

Although there are no particular limitations on the amount ofcercosporamide or salt thereof contained in the aforementionedpharmaceutical preparations, the upper limit thereof is 30 to 70% byweight, the lower limit is 1% by weight, and the preferred range is 1 to30% by weight.

Although dependent on symptoms, age, body weight, administration method,drug form and the like, the daily dosage of cercosporamide or a saltthereof for an adult has an upper limit of 100 to 1,000 mg, and a lowerlimit of 1 to 10 mg, and the preferred range is 10 to 100 mg.

The number of administrations of cercosporamide or salt thereof is onceevery several days, once a day or several times per day.

The present invention also provides a method for treating or preventinginfections comprising the administration of a pharmaceutically effectiveamount of cercosporamide or a salt thereof, and the use ofcercosporamide or a salt thereof for treating or preventing thesediseases.

5. Synthesis of Cercosporamide Derivatives

Cercosporamide produced according to the production process of thepresent invention can be used as a precursor of derivatives thereof.Although known derivatives of cercosporamide include derivatives inwhich hydroxyl groups of cercosporamide are chemically modified withether bonds, there are no particular limitations on these derivativesprovided they are able to use cercosporamide as a precursor thereof.

EXAMPLES

The present invention is explained below by way of examples thereof, butthe present invention is not limited thereto.

Example 1 Fermentation of Lachnum fuscescens SANK 19096 (FERM BP-10338)with Flask

A section of mycelia measuring about 5 mm on each side was cut out froma slant growing of strain SANK 19096 and was suspended in about 2 ml ofphysiological saline and homogenized with a glass potter. The entireamount was then aseptically inoculated into a 100 ml volumetricErlenmeyer flask containing 30 ml of the medium shown in Table 1(referred to as “Medium A”). Pre-culture was then carried out for 5 daysat 23° C. and 210 revolutions per minute (abbreviated as “rpm”) on arotary shaker.

After the resulting pre-culture was inoculated at 5% (volume/volume)(abbreviated as “v/v”) into a 500 ml volumetric Erlenmeyer flaskcontaining 80 ml of Medium A, fermentation was carried out for 7 days at26° C. and 210 rpm on a rotary shaker. HPLC analysis was then carriedout under the conditions to be described later. At that time, a peakwhich possessed an equal retention time of cercosporamide (FIG. 1) wasobserved in the culture, thereby enabling confirmation of the productionof cercosporamide by strain SANK 19096. The peak at 9.315 minutes inFIG. 1 indicates cercosporamide.

TABLE 1 Medium A: Medium Composition Sucrose 20 g Raw potato 100 gPolypepton 10 g Potassium dihydrogenphosphate 5 g Magnesium sulfateheptahydrate 2.5 g Antifoaming agent CB-442 (NOF Corp.) 100 mg Tap water1000 ml

The medium was sterilized at 121° C. for 20 minutes without adjustingpH.

HPLC analysis was carried out under the conditions indicated below.

Separation column: YMC J'sphere ODS-H80 S-4, 4.60×150 mm (YMC Co., Ltd.)

Mobile phase: acetonitrile: 0.4% triethylamine-phosphate buffer (pH 3.2)(45:55)

Flow rate: 1.0 ml/minute

Detection: Ultraviolet absorption at 225 nm

Retention time: 9.3 minutes

Example 2 Large-Scale Fermentation of Lachnum fuscescens SANK 19096(FERM BP-10338)

A section of mycelia from a slant growing of strain SANK 19096 wassuspended in about 2 ml of physiological saline and homogenized with aglass potter. The entire amount was inoculated into a 100 ml volumetricErlenmeyer flask containing 30 ml of the medium shown in Table 2(referred to as “Medium B”) followed by cultivation for 7 days at 23° C.and 210 rpm on a rotary shaker. Following completion of the culture, anequal volume of 20% glycerol was added and mixed into the culture broth.This mixture was then stored at −80° C. and used as a seed culture brothfor further use.

2 ml of the seed culture broth was inoculated into a 100 ml volumetricErlenmeyer flask containing 30 ml of Medium B. The initialpre-cultivation (first pre-cultivation) was carried out for 5 days at23° C. and 210 rpm on a rotary shaker.

Culture broth obtained in the first pre-culture (first pre-culturebroth) was inoculated at 5% (v/v) into a 2 L volumetric Erlenmeyer flaskcontaining 500 ml of Medium B followed by carrying out a second processof pre-culture (second pre-cultivation) for 4 days at 23° C. and 210 rpmon a rotary shaker.

Culture broth obtained in the second pre-culture (second pre-cultivationbroth) was inoculated at 5% (v/v) into a 60 L volumetric fermentercontaining 30 L of Medium B. A third process of pre-culturing (thirdpre-cultivation) was carried out for 2 days at an aeration rate of 30 Lper minute and a culturing temperature of 23° C. while adjusting theagitation speed so as to maintain the dissolved oxygen concentrationwithin the range of 3 to 5 ppm.

Culture broth obtained in the third pre-culture (third pre-cultivationbroth) was inoculated at 5% (v/v) into a 600 L volumetric fermentercontaining 300 L of Medium B. A fourth process of pre-culturing (fourthpre-cultivation) was carried out for 2 days at an aeration rate of 300 Lper minute and a culturing temperature of 23° C. while adjusting theagitation speed so as to maintain the dissolved oxygen concentrationwithin the range of 3 to 5 ppm.

Culture broth obtained in the fourth pre-culture (fourth pre-cultivationbroth) was inoculated at 5% (v/v) into a 6,000 L volumetric fermentercontaining 4,000 L of Medium B. Fermentation was carried out for 8 daysat a culturing temperature of 26° C. and an aeration rate of 2,000 L perminute while adjusting the agitation speed so as to maintain thedissolved oxygen concentration at 5 ppm. 200 L each of 20% sucrosesolution was added on days 4 and 5 of the fermentation. 300 L each of20% sucrose solution was further added on days 6 and 7 of thefermentation. Cercosporamide production was confirmed by the HPLC underthe conditions indicated below.

TABLE 2 Medium B: Medium Composition Glucose 40 g Potato granules(Agrawest Foods Ltd.) 20 g Polypepton 10 g Potassium dihydrogenphosphate5 g Magnesium sulfate heptahydrate 2.5 g Antifoaming agent CB-442 (NOFCorp.) 100 mg Tap water 1000 ml

The medium was sterilized at 121° C. for 20 minutes without adjustingpH.

HPLC analysis was carried out under the conditions indicated below.

Separation column: Cadenza CD-C18, 4.6φ×75 mm (Imtakt Corp.)

Mobile phase: acetonitrile: 0.02% trifluoroacetic acid (40:60); elutedwhile changing solvent composition for 8 minutes (90:10)

Flow rate: 1.0 ml/minute

Detection: Ultraviolet absorption at 225 nm

Retention time: 5.4 minutes

Example 3 Purification of Cercosporamide from Fermented Broth of Lachnumfuscescens SANK 19096 (FERM BP-10338)

The desired substance in the form of cercosporamide was confirmed by theHPLC under the conditions described in Example 2.

4,800 L of a fermented broth of Lachnum fuscescens SANK 19096 obtainedin Example 2 was filtered using Celite 545 (Celite Corp.) as afiltration aid. 2,500 L of tap water was added to 781 kg of theresulting mycelial cake including the Celite and the cake as uniformlysuspended therein. The material was then extracted by addition of 2,500L of acetone to the suspension. 4,973 L of the resulting filteredextract was applied to a 80 L Diaion HP-20 column (Mitsubishi ChemicalCorp.) equilibrated with about 500 L of a 50% aqueous acetone solution.The Diaion HP-20 column was washed with a 50% aqueous acetone solution.The liquid that passed solution through the column and washing solutionwere combined to obtain 5,500 L of solution.

75% (v/v) sulfuric acid was added to the resulting solution to adjustthe pH to 3.0 followed by extraction by addition of 3,000 L of ethylacetate. 4,218 L of solution extracted with ethyl acetate was washedwith 1,000 L of 25% brine. The ethyl acetate was removed from 3,841 L ofwashed extract by concentration in vacuo. After concentrating to 81 L invacuo, the ethyl acetate was further evaporated off using a 20 Lvolumetric rotary evaporator to obtain a concentrate.

About 10 L of the resulting concentrate was allowed to stand at 4° C. tocrystalyze cercosporamide. 4,700 g of the resulting wet crystal wasdried in vacuo to obtain 4,140 g of dry cercosporamide crystals.

Example 4 Production of Cercosporamide by Lachnum fuscescens SANK 19096(FERM BP-10338)

A section of mycelia measuring about 5 mm on each side was cut out froma slant growing of strain SANK 19096 and suspended in about 2 ml ofphysiological saline. The suspension was then homogenized with a glasspotter. The entire amount was aseptically inoculated into a 100 mlvolumetric Erlenmeyer flask containing 20 ml of Medium B. Pre-culturewas then carried out for 7 days at 23° C. and 210 rpm on a rotaryshaker. The resulting pre-cultured broth was inoculated into a 500 mlvolumetric Erlenmeyer flask containing 80 ml of Medium B having the samecomposition at 5% volume/volume (abbreviated as “v/v”) inoculum size,and the fermentation was carried out for 10 days at 26° C. on a rotaryshaker at 210 rpm. Extraction for the purpose of analysis was carriedout in the manner described below. 0.4 ml of 3 M glycinate buffer (pH3.2), 1 ml of acetone and 2 ml of n-butanol were added to 2 ml offermented broth and cercosporamide was extracted by shaking for 10minutes at room temperature. After centrifuging for 10 minutes at 3,000rpm, 2 ml of saturated brine was added to the resulting solvent layer,and washing was carried out for 10 minutes at room temperature. An oilyextract obtained by concentrating the solvent layer in vacuo wasdissolved in 0.2 ml of a solution of dimethylsulfoxide and methanol(7:3). This extract solution was subjected to HPLC under the conditionsdescribed below. At this time, a peak having the same retention time ascercosporamide used as a control (retention time: 20.7 minutes) wasobserved.

Medium Composition Glucose 40 g Potato granules (Agrawest Foods Ltd.) 20g Polypepton 10 g Potassium dihydrogenphosphate 5 g Magnesium sulfateheptahydrate 2.5 g Antifoaming agent CB-442 (NOF Corp.) 500 mg Tap water1000 ml

The medium was sterilized for 20 minutes at 121° C. without adjustingpH.

HPLC analysis was carried out under the conditions indicated below.

Column: Symmetry C-18, 4.6φ×150 mm (Waters Corp.)

Mobile phase: acetonitrile: 0.3% triethylamine phosphate buffer (pH 3.2)(5:95); eluted while changing solvent composition for 28 minutes (90:10)

Flow rate: 1.0 ml/minute

Detection: Ultraviolet absorption at 225 nm

Example 5 Production of Cercosporamide by Lachnum calycioides SANK 12497(FERM BP-10636)

A section of mycelia measuring about 5 mm on each side was cut from aslant growing of strain SANK 12497 followed by suspending in about 2 mlof physiological saline and homogenizing with a glass potter. Thesubsequent fermentation, extraction and analysis were carried out in thesame manner as Example 4. By HPLC analysis, a peak coinciding with theretention time of cercosporamide was observed in the fermented broth(retention time: 20.7 minutes).

Example 6 Production of Cercosporamide by Lachnum caesaliatum SANK 10906(FERM BP-10634)

A section of mycelia measuring about 5 mm on each side was cut from aslant growing of strain SANK 10906 followed by suspending in about 2 mlof physiological saline and homogenizing using a glass potter. Thesubsequent fermentation, extraction and analysis were carried out in thesame manner as Example 4. By HPLC analysis, a peak coinciding with theretention time of cercosporamide was observed in the fermented broth(retention time: 20.7 minutes), thereby confirming that strain SANK10906 produces cercosporamide.

Example 7 Production of Cercosporamide by Pseudaegerita websteri SANK11006 (FERM BP-10635)

A section of mycelia measuring about 5 mm on each side was cut from aslant growing of strain SANK 11006 followed by aseptically inoculatinginto a 100 ml volumetric Erlenmeyer flask containing 20 ml of medium.The subsequent fermentation, extraction and analysis were carried out inthe same manner as Example 4. By HPLC analysis, a peak coinciding withthe retention time of cercosporamide was observed in the fermented broth(retention time: 20.7 minutes), thereby confirming that strain SANK11006 produces cercosporamide.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a chromatogram obtained during HPLC analysis ofcercosporamide obtained from a fermented broth of Lachnum fuscescensSANK 19096 (FERM BP-10338).

INDUSTRIAL APPLICABILITY

Cercosporamide can be produced according to the present invention, andcercosporamide produced according to said process can itself be used asan active ingredient of a pharmaceutical. In addition, thiscercosporamide can also be used as a precursor of a cercosporamidederivative.

1. A process for producing cercosporamide comprising: culturing a fungusbelonging to the genus Lachnum and recovering cercosporamide from theculture thereof, wherein the fungus belonging to the genus Lachnum is atleast one fungus selected from the group consisting of Lachnumfuscescens SANK 19096, Lachnum calycioides SANK 12497, and Lachnumcaesaliatum SANK
 10906. 2. A process for producing cercosporamidecomprising culturing Lachnum fuscescens SANK 19096 and recoveringcercosporamide from the culture thereof.
 3. A process for producingcercosporamide comprising culturing Lachnum calycioides SANK 12497 andrecovering cercosporamide from the culture thereof.
 4. A process forproducing cercosporamide comprising culturing Lachnum caesaliatum SANK10906 and recovering cercosporamide from the culture thereof.